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Two papers in the September 2 Nature Cell Biology present complementary evidence that tau condenses into a patchwork of rafts that coat microtubules. These patches share some properties with phase-separated liquid droplets of tau that have been described recently. Zdenek Lansky and Marcus Braun of the Czech Academy of Sciences, Prague West, and Amayra Hernández-Vega at the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, led one of the studies, while husband-and-wife team Richard McKenney and Kassandra Ori-McKenney of the University of California, Davis, led the other. Both groups report that these islands of tau regulate movement of microtubule-associated motors and fend off enzymes that would cut up the tubulin strands. Together, the papers suggest that tau condensation plays a major role in its physiological function, and that it is key to microtubule regulation.

Tau condenses in monolayer patches on microtubules.

Patches regulate movement of molecular motors and action of enzymes.

Loss of healthy tau condensation could lead to microtubule degradation.

“These studies open up a new way of thinking about tau, and recognize it as a liquid, condensed phase in the physiological context,” said Susanne Wegmann, DZNE, Berlin.

Benjamin Wolozin, Boston University School of Medicine, agreed. “These studies extend our understanding of the importance of phase separation in biology, and might ultimately impact on our understanding of the pathophysiology of tauopathies,” he wrote to Alzforum.

Tau is a microtubule-associated protein (MAP) known to stabilize microtubules, regulate traffic of other MAPs, and protect against tubulin proteolysis (Drechsel et al., 1992; Chaudhary et al., 2018; Qiang et al., 2006). It was thought to bind microtubules as individual molecules, although some reports suggested tau self-associates along the microtubule and forms intermittent solid patches (Ackmann et al., 2000; Dixit et al., 2008). Two years ago, reports began to surface that tau undergoes liquid-liquid phase separation—condensing into droplets in solution (May 2017 news; Jul 2017 news; Aug 2017 news). Does this play a role in normal, healthy function and could it be important for microtubule biology?

Strung Along. On a hippocampal neuron from a mouse (left), patches of tau (green) line up on along microtubules (red). Magnification at right. [Courtesy of Tan et al., 2019.]

To find out, both groups of researchers used total internal reflection fluorescence (TIRF) microscopy to watch how tau behaved on microtubules assembled in the lab. In this technique, a laser focuses on one tiny area of the specimen at a time, minimizing background fluorescence and improving spatial resolution down to the single-molecule level.

Through this technique, both groups saw that tau bound the microtubule surface in two ways, either diffusely as individual molecules or in dynamic islands of condensed tau proteins that dotted the length of microtubules. The islands—cohesive, single-layer coatings of tau—were of uniform density and grew and shrank from their outer boundaries. Islands fused when they met along the microtubule length. Their formation required a minimum concentration of soluble tau.

Within these islands, tau’s mobility slowed relative to tau bound to microtubules as single molecules. Island tau exchanged more slowly with free tau and took longer to detach from microtubules once free tau was removed from solution. The findings suggested that within these condensed patches, tau molecules interact with one another.

Working with Braun, co-first authors Valerie Siahaan and Jochen Krattenmacher at the Czech Academy of Sciences found that while the kinesin-1 motor could easily move between islands, it fell off microtubules as soon as it encountered an island. On the other hand, the molecular motor Kip3 moved through islands, albeit at a slower velocity than it traveled between them. Condensed tau blocked microtubule digestion by the hydrolase katanin.

Writing in the McKenney paper, first author Ruensern Tan reported that the islands allowed movement of dynein complexes, but were impervious to the microtubule-severing enzyme spastin. Together, the data suggest that tau islands form selectively permissible barriers that spatially regulate the location and action of other microtubule-associated proteins.

As reported in the McKenney paper, the tau-tau interactions on microtubules are similar to, though distinct from, those occurring when tau condenses in the liquid phase. Both condensates are reversible, depend on tau concentration, can fuse with neighboring droplets/islands, and can be dissolved by the alcohol 1,6 hexanediol. However, the single-layer tau condensation on microtubules does not take on a gel-like consistency, nor does it exchange as quickly with tau in solution. This suggests that the microtubules have a stabilizing effect on tau, say the authors.

The findings have raised many questions. Wegmann wondered how physiological and pathological phosphorylation affects island dynamics. Braun is examining if tau mutations might do the same. Hernández-Vega speculates that other microtubule proteins may form similar patches that spatially sort proteins and regulate transport.

TIRF microscopy is a powerful technique, commented Khalid Iqbal, New York State Institute for Basic Research, Staten Island, New York. He cautioned that it remains to be seen whether tau islands exist in vivo. Though the authors found tau patches on neurons in mice (see image above), all the mechanistic experiments were done in vitro.

“One of the biggest take-home messages of our work is that the self-interaction of tau molecules is not just a disease-specific phenomenon, but rather may be a common molecular process for tau to perform its normal cellular functions,” wrote the McKenneys to Alzforum. “The loss of normal condensate dynamics could be detrimental to cellular control over these processes.”

That could have implications for health. Since a minimum concentration of soluble tau is required for islands to form, neurofibrillary tangles or other aggregates that sequester the protein could limit island formation. “Microtubules would then fall apart because they would be eaten up by enzymes such as katanin,” said Braun.

“In addition to the important cell biological insights reported in these two papers, they imply the necessity for caution as the field moves toward trials for AD and other tauopathies intended to reduce total tau,” wrote Kenneth Kosik, University of California, Santa Barbara. “Therapeutic strategies that reduce total tau synthesis, such as the use of antisense oligonucleotides, will affect pools of tau on microtubules and consequently microtubule function in ways that are not easily predictable.”—Gwyneth Dickey Zakaib

Comments

Both studies elegantly identify novel interactions between tau and microtubules (MTs) in a physiological setting, either in regulating tau condensation or in protecting MTs from depolymerizing. I have been reminded of a paper from the Safinya lab on the role of tau on MT architecture focusing on the axon initial segment (Chung et al., 2016). This work also focused on the projection domain of tau (see → Fig 3 of Tan et al.). It would be interesting to determine, as a follow-up of McKenney's work, how/whether microtubules gate tau condensation in the axon initial segment, considering that this is the compartment in which action potentials are generated.

How are these processes affected by pathological tau? In a disease context it has been shown by us that pathological tau impairs action potential generation (neuronal excitability) in a microtubule-dependent manner (Hatch et al., 2017). Considering that the MT caliber differs between axons and dendrites, it might also be worthwhile to assess the role of dendritic tau, again under physiological and pathological conditions. By extension, it would be interesting to find how MAP2 becomes organized in the dendrite, and what the changes are with development.

These new tau papers advance our knowledge of tau protein function at a mechanistic level. Previous work showed that tau is an RNA-binding protein capable of undergoing liquid-liquid phase separation (LLPS) (Zhang et al., 2017) and contextualized tau with other intrinsically disordered RNA-binding proteins capable of LLPS that can transition to solid inclusions. Subsequently, several other groups extended these data (Wegmann et al., 2018; Hernández-Vega et al., 2017; Ambadipudi et al., 2017). Underlying all these papers are two questions: (1) Is there a physiological role for tau phase separation? And (2) Is tau LLPS on the pathway to pathological tau aggregation? The heuristic approaches to these two questions often overlap.

Ackmann et al. (2000) pointed out that tau binding to microtubules is biphasic with a nonsaturable second phase suggestive of tau-tau interactive binding induced by the polyanionic C-terminal domain of tubulin. This would be consistent with the self-assembly of tau in association with other polyanions. Further experiments with Alexa-labeled tau decorated taxol-stabilized microtubules in patches of three to 20 labeled tau molecules that extended up to 1.2 μm (Dixit et al., 2008). More recently clustering of tau has been described in terms of LLPS. When tau droplets were aged in vitro, a thioflavin signal emerged suggesting a conformational change that resembled the fibril of the neurofibrillary tangle (Zhang et al., 2017; Wegmann et al., 2018). This property of tau coacervation was further pinned down to the microtubule-binding repeats, the amyloid-promoting elements of tau (Ambadipudi et al., 2017); however, other studies including the paper here by Tan et al. indicate a complex relationship of the tau domain structure to tau condensation. However, within tightly packed tau condensates, tau can retain its normal conformation as shown by electron spin resonance (ESR) of tau droplets (Zhang et al., 2017). Tau has also been shown to affect several microtubule regulatory proteins including severing activity (Vale, 1991; Qiang et al., 2006; Yu et al., 2008) and has important effects on microtubule motors (Dixit et al., 2008; Vershinin et al., 2007; Monroy et al., 2018; Seitz et al., 2002).

Tan et al. have performed an elegant study that begins with the demonstration that tau initially binds diffusely along the entire microtubule (MT) lattice, but over time expansion of denser regions occurs gated by the nucleotide state of the MT lattice, probably corresponding to the GMP-CPP versus GDP state of the microtubules. This observation allowed the authors to hypothesize that the spacing between tubulin dimers regulates the ability of tau to undergo condensation on the lattice. These condensates passed several tests for LLPS—the ability to FRAP (fluorescence recovery after photobleaching), fuse, and dissolve on addition of 1,6-hexanediol (1,6-HD). From these in vitro studies they transitioned to live cell experiments and reached the conclusion that tau condensates along the microtubule and that its specific interactions with cargo can adjust the velocity and run lengths of retrograde traffic. These studies open a rich territory to find potential effects of pathological tau mutations on condensate formation and on the interactions of condensates with retrograde motors and cargo.

The companion paper by Siahaan et al. emphasizes the control of microtubule-severing enzymes by tau, and by implication, microtubule destabilization and axonal degeneration as a result of tau mislocalization. They describe the association of tau with microtubules as cooperatively forming cohesive islands that that are kinetically distinct from tau molecules that individually diffuse on microtubules. Tau islands on microtubules halted the processive movement of kinesin motors and prevented the activity of microtubule-severing enzyme katanin. Tau conferred microtubule protection from severing due to cohesion between the cooperatively binding tau molecules that make up the islands. As expected based on the properties of phase states, which are maintained by weak interactions, small changes in concentration or charge will alter the balance between these two kinetically distinct pools of tau. In fact, the authors showed that the rate of tau unbinding from the islands increased with increasing tau concentration in solution.

In addition to the important cell biological insights reported in these two papers, they imply the necessity for caution as the field moves toward trials for AD and other tauopathies intended to reduce total tau. Therapeutic strategies that reduce total tau synthesis, such as the use of antisense oligonucleotides, will affect pools of tau on microtubules and consequently microtubule function in ways that are not easily predictable. The implementation of such interventions before we have determined how the dynamical states of tau lead to neurofibrillary tangles is premature.

These papers by Siahaan et al. and Tan et al. add to a growing body of work emphasizing the importance of phase separation in biology, and the specific importance of this work in tau biology. Previous work by the Hyman laboratory demonstrated the condensation and phase separation of tau around microtubules (Hernández-Vega et al., 2017). The current work extends our understanding greatly, and in multiple directions.

The first striking point is that the experiments can use very low, highly relevant physiological levels of tau (20 nM); this might be because the condensation of tau around microtubules allows a ready nidus for concentrating the protein. These results contrast with phase separation of tau alone (with RNA), which requires micromolar amounts.

Other points are equally interesting. The McKenney group identifies regions critical for tau condensation that differ from that observed with tau alone (projection domain vs. microtubule-binding domain). The group also shows how condensation of tau regulates particular microtubule functions and reveals a fascinating selectivity of tau condensation for curved microtubules. Meanwhile, the manuscript by the Braun/Lansky/Hernandez-Vega group shows domains of tau on microtubules that exhibit differential dynamic movement.

These studies begin to extend our understanding of the importance of phase separation in biology, and might ultimately impact on our understanding of the pathophysiology of tauopathies.